ß-Catenin is required for Ron receptor-induced mammary tumorigenesis.

Our previous studies demonstrated that selective overexpression of the Ron receptor tyrosine kinase in the murine mammary epithelium leads to mammary tumor formation. Biochemical analysis of mammary tumor lysates showed that Ron overexpression was associated with increases in ß-catenin expression and tyrosine phosphorylation. ß-Catenin has also been shown to be regulated through tyrosine phosphorylation by the receptor tyrosine kinases Met, Fer and Fyn. However, the molecular and physiological roles of ß-catenin and ß-catenin tyrosine phosphorylation downstream of Ron are not known. To investigate this association, we show that Ron and ß-catenin are coordinately elevated in human breast cancers. Our data also demonstrate that activation of Ron, through ligand binding by hepatocyte growth factor-like protein (HGFL), induces the tyrosine phosphorylation of ß-catenin, primarily on tyrosine residues Tyr 654 and Tyr 670. In addition, HGFL-mediated Ron activation induces both ß-catenin nuclear localization and transcriptional activity, with Tyr 654 and Tyr 670 residues of ß-catenin being critical for these processes. We also demonstrate that a knockdown of Ron in breast cancer cell lines leads to a loss of HGFL-induced ß-catenin-dependent transcriptional activation and cell growth, which can be rescued by activation of canonical Wnt/ß-catenin signaling. Moreover, we show that HGFL-dependent Ron activation mediates upregulation of the ß-catenin target genes cyclin D1 and c-myc, and that expression of these target genes in breast cancer cells is decreased following inhibition of Ron and/or ß-catenin. Finally, we show that genetic ablation of ß-catenin in Ron-expressing breast cancer cells decreases cellular proliferation in vitro, as well as mammary tumor growth and metastasis, following orthotopic transplantation into the mammary fat pad. Together, our data suggest that ß-catenin is a crucial downstream regulator of Ron receptor activation and is an important mediator of mammary tumorigenesis.

Dietary calcium does not affect prostate tumor progression in LPB-Tag transgenic mice.

High dietary calcium has been shown in epidemiological studies to be a risk factor for prostate cancer, and it has been postulated that this effect is secondary to calcium induced modulation of the vitamin D axis. In this study, we used LPB-Tag transgenic mice on the CD1 background to examine the impact of dietary calcium on prostate tumor progression. CD1-LPB-Tag mice predictably develop autochthonous, hormone-responsive prostate tumors by 3 months of age. Age matched transgenic and non-transgenic littermates were weaned onto high (2%) or low (0.2%) calcium diets and mice were sacrificed at 5, 7, and 9 weeks of age. The entire urogenital complex was excised, weighed, and processed for histology. There was no significant effect of dietary calcium on tumor weight or on the time course of tumor progression, as monitored using a modified Gleason grade (MGS). Serum calcium was maintained in the normal range in mice on the low and high calcium diet throughout the study. Circulating 1,25(OH)(2)D(3) was elevated by low dietary calcium in 5-week-old mice, but not in older animals. In summary, neither development nor progression of prostate tumors in LPB-Tag mice was accelerated by high dietary calcium.

[Modified Heidelberg Retinal Tomograph HRT. Initial results of in vivo presentation of corneal structures]. / Der modifizierte Heidelberg-Retina-Tomograph HRT. Erste Ergebnisse einer In-vivo-Darstellung von kornealen Strukturen.

BACKGROUND: At present, confocal tandem scanning microscopes with halogen or mercury lamps are used to depict all corneal structures in vivo, e.g., before and after PRK or LASIK. Insufficient imaging quality and irregular corneal illumination are the main problems for automatic quantitative evaluation of the keratocyte density when applying this instrument. A high correction is required for correcting the background irregularities of pictures. Our aim was to find out whether it is possible to change the Heidelberg retina tomograph (HRT) into a high-resolution digital laser scanning microscope for the visualization of anterior segments of the eye, coupled with a special evaluation software. MATERIAL AND METHOD: We developed a lens adapter for the HRT that focusses the laser beam onto the cornea by combining with an external, computer-controlled hydraulic z-scan sledge. By using a programmable adaptation for the external stepmotor on the z-scan sledge in combination with all internal control functions and patient data, it is possible to create a digital confocal laser scanning microscope with retention of all the original HRT functions. For evaluation of the corneal images and automatic count of keratocytes, we used special 3D and Chemotaxis software. RESULTS: First investigations show a regular illumination of all corneal structures as the epithelium, endothelium, and keratocytes. The hydraulic z-scan allowed a precise shift of the focus through the cornea to take series of images for the evaluation of the keratocyte profile and 3D reconstruction of all corneal structures.

Functions of 1alpha,25-dihydroxyvitamin D(3) in mammary gland: from normal development to breast cancer.

This review examines the role of 1alpha,25(OH)(2)D(3) (1,25D) and the vitamin D(3) receptor in growth regulation of normal and transformed mammary epithelial cells. 1,25D exerts both anti-proliferative and pro-apoptotic functions in transformed mammary cells such as MCF-7. The anti-proliferative effects of 1,25D have been linked to suppression of growth stimulatory signals and potentiation of growth inhibitory signals, which lead to changes in cell cycle regulators such as p21, p27, cyclins and Rb. The pro-apoptotic effects of 1,25D involve alterations in the relative ratios of the bcl-2 family members which regulate mitochondrial integrity. In MCF-7 human breast cancer cells, 1,25D mediated apoptosis is associated with translocation of the pro-apoptotic protein Bax to the mitochondria, generation of reactive oxygen species, dissipation of the mitochondrial membrane potential and release of cytochrome c. These mitochondrial events trigger apoptosis in a caspase-independent manner, since caspase inhibitors do not rescue 1,25D treated cells from death. The potential role of 1,25D in growth and differentiation of normal mammary epithelial cells has been examined in VDR null mice. Initial data indicates a significant decrease in ductal differentiation in VDR null mice compared to age matched wild type mice, reflected as an increased number of undifferentiated terminal end buds in the VDR null mouse. These data suggest that 1,25D promotes differentiation during early mammary gland development. In summary, our studies suggest an expanding role for the vitamin D(3) endocrine system in control of proliferation, differentiation and apoptosis of mammary epithelial cells.

vrrB, a hypervariable open reading frame in Bacillus anthracis.

Bacillus anthracis appears to be the most molecularly homogeneous bacterial species known. Extensive surveys of worldwide isolates have revealed vanishingly small amounts of genomic variation. The biological importance of the resting-stage spore may lead to very low evolutionary rates and, perhaps, to the lack of potentially adaptive genetic variation. In contrast to the overall homogeneity, some gene coding regions contain hypervariability that is translated into protein variation. During marker analysis of diverse strains, we have discovered a novel ca. 750-nucleotide open reading frame (ORF) that contains in-frame, variable-number tandem-repeat sequences. Four distinct variable regions exist within vrrB, giving rise to 11 distinct alleles in eight different length categories among B. anthracis strains. This ORF putatively codes for a 241- to 265-amino-acid protein, rich in glutamine (13.2%), glycine (23.4%), and histidine (23.0%). The variable-region amino acids of the vrrB ORF are strongly hydrophilic. Coupled with putative transmembrane domains flanking the variable regions, this suggests a membrane-anchored cytosolic or extracellular location for the putative protein. Sequence analysis of the complete ORFs from three Bacillus cereus strains shows maintenance of the ORF across species boundaries, including strong conservation of the amino acid sequence and the capacity to vary among strains. The presence of 11 different alleles of the vrrB locus is in stark contrast to the near homogeneity of B. anthracis. Evolution of hypervariable genes can negate the lack of genetic variability in species such as B. anthracis and provide select rapid evolution in other more variable species.

Molecular diversity in Bacillus anthracis.

Molecular typing of Bacillus anthracis has been extremely difficult due to the lack of polymorphic DNA markers. We have identified nine novel variable number tandemly repeated loci from previously known amplified fragment length polymorphism markers or from the DNA sequence. In combination with the previously known vrrA locus, these markers provide discrimination power to genetically characterize B. anthracis isolates. The variable number tandem repeat (VNTR) loci are found in both gene coding (genic) and non-coding (non-genic) regions. The genic differences are 'in frame' and result in additions or deletion of amino acids to the predicted proteins. Due the rarity of molecular differences, the VNTR changes represent a significant portion of the genetic variation found within B. anthracis. This variation could represent an important adaptive mechanism. Marker similarity and differences among diverse isolates have identified seven major diversity groups that may represent the only world-wide B. anthracis clones. The lineages reconstructed using these data may reflect the dispersal and evolution of this pathogen.

Confocal scanning infrared laser ophthalmoscopy for indocyanine green angiography.

PURPOSE: We used indocyanine green to study wavelength-optimized confocal scanning infrared laser angiography in patients with retinal and choroidal disease. METHODS: A confocal scanning laser ophthalmoscope with an excitation wavelength of 795 nm was operated both in tight and wide confocal imaging modes. We examined 77 subjects with and without retinal and choroidal disease (including diabetic retinopathy, age-related macular degeneration, and subretinal neovascularization). RESULTS: The scanning laser ophthalmoscope allowed acquisition of images, in the wide confocal imaging mode, of the retinal circulation and late leakage sites without late injections of dye to outline the retinal vasculature. In the tight confocal imaging mode, optical subtraction of the light contribution of the retinal circulation allowed examination of the choroidal circulation, and vice versa. The wide confocal mode appears equivalent to other scanning laser ophthalmoscopes in recording images from retinal and choroidal layers. CONCLUSIONS: There are three differences between the confocal scanning laser ophthalmoscope and conventional instruments. First, the late images allow excellent visualization of the retinal circulation without a landmark injection. Second, confocal imaging allows optical subtraction of retinal circulation when focusing on the choroid and vice versa. Third, the instrument acquires and processes all data digitally, is personal computer-based, is compact, operates with a mouse-driven graphical user interface, and allows easy data exchange with conventional software. With further modifications in software and hardware, this device offers the possibility of producing a three-dimensional map of the retinal and choroidal vasculature.

Behavioral health at-risk contracting--a rate development and financial reporting guide.

The process of developing rates for behavioral capitation contracts can seem mysterious and intimidating. The following article explains several key features of the method used to develop capitation rates. These include: (1) a basic understanding of the mechanics of rate calculation; (2) awareness of the variables to be considered and assumptions to be made; (3) a source of information to use as a basis for these assumptions; and (4) a system to collect detailed actual experience data.

[Examinations of the cornea and anterior chamber angle region with a laser tomographic scanner (LTS)]. / Untersuchungen der Hornhaut und der Kammerwinkelregion mit dem Laser Tomographic Scanner (LTS).

The Laser Tomographic Scanner LTS based on laser scanning and confocal detection is a system to generate and measure optical section images. The periphery of the cornea and the anterior chamber angle region is shown in the horizontal section image with high image quality and high signal-to-noise ratio. 130 patients and 163 eyes were examined in this way. First experience shows possible applications for special clinical situations. Using horizontal optical section images, a procedure is generated to measure the anterior chamber angle without contact or local anesthesia with a reproducibility of 2 degrees. Horizontal section images clearly demonstrate changes of anterior chamber angle depth following e.c. cataract extraction and implantation of posterior chamber IOLs. Using the generated measurement procedure, these changes could be quantified.

Reproducibility of topographic measurements of the optic nerve head with laser tomographic scanning.

Topographic analysis and measurement of the optic nerve head is important for the diagnosis and follow-up of glaucoma. To quantify structures of the optic nerve head the new technique of laser tomographic scanning was used. A laser beam was focused onto the surface of the optic nerve head and the reflected light was detected in a confocal detection unit. The consequent change of focus produced a tomographic scanning series and allowed measurement of three-dimensional structures. To analyze the reproducibility of optic cup measurements the authors did ten recordings of one eye of eight normal volunteers. The mean standard deviation of the measurements was +/- 0.015 mm3 and the mean coefficient of variation was 9.5%. Confocal laser tomographic scanning is a safe, effective, convenient method to measure and document the topography of the optic nerve head and should be a valuable technique for follow-up of glaucoma patients.

[3-dimensional biomorphometry of the papilla using a laser tomography scanning procedure--initial experiences with pathologic papillar findings]. / Zur dreidimensionalen Biomorphometrie der Papille mit dem Lasertomographicscanningverfahren--erste Erfahrungen an pathologischen Papillenbefunden.

Three-dimensional topographical analysis of the optic nerve head and the parapapillary region was performed with a confocal laser tomographic scanner. Four patients with various disorders of the optic nerve head (glaucoma, optic disk pit, morning glory syndrome and a parapapillary neoplasm) were investigated as examples of application of the method in a clinical setting.

Conducting a financial analysis of capitation arrangements.

There is no doubt that capitation contracts are risky business. There are many issues to consider carefully that have implications in every area of the healthcare industry. However, there are many additional issues that should be considered such as hospital facility capacity, incremental cost of services, physician relations, and emergency services. The contract structure and other legal issues should also be thoroughly discussed. While there will always be an element of risk in capitation arrangements, the only hope of that risk being at a reasonable and manageable level is to be prepared both financially and operationally if and when market conditions force you to consider an at-risk healthcare delivery system.

Experimental modification of postnatal cerebellar granule cell migration in vitro.

Histotypic migration of [3H]thymidine pulse-labeled granule cell neurons in cerebellar folium explants was monitored in the presence of antibodies to cell adhesion molecules and quantified by automatic image analysis. When explants were cultured in the presence of monovalent antibody fragments to cell adhesion molecules L1 and N-CAM, an inhibition of cell migration of 33.3 +/- 4.4% and 13.9 +/- 2.1%, respectively, was observed. In the presence of an equimolar mixture of monovalent antibody fragments to L1 antigen and N-CAM no additive effects in inhibition of cell migration were seen. Antibodies to the L2 carbohydrate epitope which is common to L1, N-CAM and other cell surface glycoproteins showed a similarly small effect on cell migration as antibodies to N-CAM. Monoclonal antibodies to cell surface antigen M2 and polyclonal antibodies to mouse liver membranes reacting with the surface of all cerebellar cell types did not alter the migratory behavior of granule cells. Cultivation of explants in the presence of neuraminidase, ganglioside binding toxins, as well as glycosaminoglycans and glycosaminoglycan degrading enzymes, also did not modify the extent of cell migration under the culture conditions used.

Modulation of granule cell migration by a glia-derived protein.

Cultured explants from early postnatal mouse cerebellum were used to examine the influence of a 43-kDa glia-derived neurite-promoting factor (GdNPF) on the migration of [3H]thymidine-labeled granule cell neurons. GdNPF, which is a potent serine protease inhibitor, significantly reduced the extent of granule cell migration in a dose-dependent manner. This effect could be neutralized by addition of thrombin, which binds GdNPF. Other protease inhibitors such as aprotinin, hirudin, soybean trypsin inhibitor, leupeptin, 6-aminocaproic acid, and D-Phe-Pro-ArgCH2Cl do not show this inhibitory effect. These results demonstrate that a glia-derived protein can regulate the migration of postmitotic neurons, an important cellular event in the development of the nervous system.

Quantitative evaluation of heterochromatin in epithelial atypia by an image processing method.

Digital image analysis as a method enabling quantitative description of microscopical images is especially important in studying cellular atypia. However, by using that method for characterizing the complex chromatin structure and its changes during atypia considerable difficulties arise. Defining substructures of chromatin images we have developed new method for description chromatin structure based on locally adaptive thresholding. The results obtained suggest that typical for atypia are changes in the size, optical density and distribution of the high optical density regions (heterochromatin) identified within the cell nuclei.

Quantitative description of chromatin structure during neoplasia by the method of image processing.

Nuclear chromatin is visualized by light microscopy as a mosaic of interchanging regions of low and high optical density (O.D.). The regions of high O.D. are well-defined as chromatin particles; features characterizing these particles enable the description of chromatin structure and the recognition of its changes during neoplasia. This paper presents a method of feature extraction by means of digital image analysis, based on a localization algorithm with locally adaptive thresholding. Although the chromatin particles varied greatly in their O.D., the algorithm enabled the identification of significant numbers of particles, which is essential in characterizing the complex architecture of chromatin. The results obtained by studying neoplastic nuclei and nuclei from control tissue suggest that the appearance of an additional class of chromatin particles, defined by their localization and optical density, is typical of chromatin changes during neoplasia.

Segmentation of cell nuclei in tissue section analysis.

Image segmentation is a critical step in digital picture analysis, especially for that of tissue sections. As the morphology of the cell nuclei provides important biological information, their segmentation is of particular interest. The known segmentation methods are not adequate for segmenting cell nuclei of tissue sections; the reason for this lies in the optical properties of their images. We have developed new gradient methods of segmentation of previously presegmented images by taking these properties into account and by using the approximately circular shape of the cell nuclei as a priori information. In our first technique, the segment method, the images of the nuclei are divided into eight segments, special gradient filters being defined for each segment. This has enabled us to improve the gradient image. After searching for local maxima, the contours of nuclei can be found. In the second method, the method of transformation into the polar coordinate system (PCS), the a priori information serves to define a circular direction field for gradient computation and contour finding. In contrast with the first method, which offers a rapid, general idea about the nuclear shape, the PCS method permits precise segmentation and morphological analysis of the cell nuclei.